PB0352 - Secretion of transforming growth factor β1 from megakaryocytes is mediated by autophagy signaling pathways

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Isabelle C. Becker, PhD

Postdoctoral Fellow
Boston Children's Hospital
Boston, Massachusetts, United States

Co-Author(s)

JL

Joshua Lykins

Graduate Research Assistant
University of Kentucky
Lexington, Kentucky, United States
Maria Barrachina, PhD

Post-Doctoral Research Fellow
Harvard Medical School, Boston Children's Hospital
Boston, Massachusetts, United States
Harvey G. Roweth, PhD

Post-Doctoral Researcher
Harvard Medical School, Brigham and Women's Hospital
BOSTON, Massachusetts, United States
BN

Bernhard Nieswandt

Professor
Institute of Experimental Biomedicine - Chair I, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Biomaging, University of Würzburg, Germany
Würzburg, Bayern, Germany
SW

Sidney W. Whiteheart

University Research Professor
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA
Lexington, Kentucky, United States
Kellie Machlus, PhD

Assistant Professor
Boston Children's Hospital
Boston, Massachusetts, United States
JI

Joseph Italiano

Associate Professor
Boston Children's Hospital
Boston, Massachusetts, United States

Background: Recent advances in single cell omics have identified that megakaryocytes (MKs) can function beyond platelet production by contributing to the maintenance of bone marrow homeostasis and immunity. Clonal hematopoiesis causes an accumulation of immature MKs in the bone marrow, which in turn instigates myeloproliferative disorders such as myelofibrosis. Myelofibrosis progression has been associated with an increased release of profibrotic cytokines such as transforming growth factor β1 (TGFβ1) from MKs into the bone marrow. While the underlying mechanisms remain elusive, unconventional trafficking mechanisms involving the cytoskeletal regulator RhoA and the endosomal GTPase Arf6 have been implicated in other cell types.

Aims: The aim of the study was to identify whether TGFβ1 release from MKs is autophagy-dependent, and whether underlying signaling pathways can be targeted to alter cytokine secretion in vivo.

Methods: MK secretion was analyzed in the presence of autophagy and cytoskeletal inhibitors using cytokine arrays and immunosorbent assays. Immunofluorescence staining for TGFβ1 and secretory regulators was performed on native and in vitro-matured MKs. Alterations in hematopoietic progenitor populations in mouse models with platelet secretion defects were assessed in cryosections by immunofluorescence and by flow cytometry.

Results: Treatment of MKs with autophagy or RhoA pathway inhibitors markedly reduced TGFβ1 secretion in vitro without affecting secretion of other soluble factors and was associated with an intracellular accumulation of TGFβ1 and autophagosomes. Colocalization of TGFβ1 with autophagosomes in native MKs correlated with Arf6 expression patterns. Moreover, myelofibrosis was identified in mice selectively lacking Arf6 in platelets and MKs, while other mouse models with platelet secretory defects did not exhibit altered marrow morphology.

Conclusion(s): Our data suggest that TGFβ1 trafficking is uncoupled from conventional granule secretion, but dependent on the RhoA and Arf6 signaling pathways that regulate secretory autophagy. Future studies will evaluate MK autophagy as a possible therapeutic target in myelofibrosis.